1. Field of the Invention
The present invention discloses xanthene-based, carboxamide-substituted dyes of the general formula (I) as well as the preparation, activation, and use of said dyes.

The dyes according to formula (I) have very good spectral properties, such as the position of the absorbance and emission bands, high extinction coefficient, high fluorescent quantum yields, and photostability. The disadvantage of lactone or lactam formation, which is associated with the presence of carboxylic acid at the 2nd position of the “lower” aromatic ring, is prevented by the conversion of said carboxylic acid group into a secondary amide.
The dyes of this invention possess considerable advantage over their previously described xanthene dyes. In particular, their fluorescence yields are typically higher than those of other dyes having compatible spectra, including fluorescein, Cy-2, tetramethylrhodamine, Cy-3, and Texas Red. In addition, the dyes of this invention exhibit enhanced resistance to quenching upon protein conjugation, and protein conjugates with the dyes of the invention typically possess substantially higher fluorescence yields than that achieved with most commercially available fluorescent dyes, including AlexaFluor dyes. Also, the dyes of this invention are substantially more water-soluble than dyes without an amido-sulfoalkyl group.
2. Description of the Background
Organic fluorescent compounds, also known as dyes, are widely used as sensitive detection reagents in biological systems. Xanthene-type dyes are among the most frequently organic fluorescent compounds used as detection reagents due to their very good spectral properties and photostability. The dyes having a very high fluorescence quantum yield are especially important since the fluorescence enables the labeled analyte to be detected at very high sensitivity.
Many xanthene-type dyes possess a carbonyl group at the o-position of the “lower” aromatic ring, which causes the formation of a colorless lactone under some physiological conditions. The lactone is colorless and non-fluorescent. Thus, the labeled analyte cannot be detected by means of fluorescent spectroscopy.

The traditional way to covalently attach a fluorescent dye to a biomolecule is through a reaction of a primary amine group of the biomolecule with an activated ester, for example dyes of NHS ester. One way of rendering the formation of non-fluorescent lactone is to use the carboxylic group o-position of the “lower” aromatic ring for covalent attachment of a fluorescent dye to the biomolecule. However, this reaction produces a primary amide, which immediately rearranges into a lactam according to Scheme 1.
The lactam is colorless and non-fluorescent under physiologically relevant conditions. Thus, the labeled analyte cannot be detected by means of fluorescent spectroscopy.
WO 02/055512 and US 2006/0154251 A disclose the preparation of amide derivatives of fluorescein and rhodamine dyes, which comprise the conversion of carboxylic acids into activated esters followed by reaction of said activated esters with a secondary amine under reflux conditions to form secondary amides. Even though the disclosed secondary amides do not form non-fluorescent lactone or rearrange into lactam, they possess several major shortcomings. First, the disclosed secondary amides are known to undergo hydrolysis under basic conditions (Boyarskiy, V. P. et al., Chem. Eur. J., 14:1784, 2008), resulting in dissociation of a fluorescent dye from an analyte, so the dissociated fluorescent dye will be detected by means of fluorescent spectroscopy and not the labeled biomolecule of interest. Second, conversion of a carboxylic acid into a secondary amide makes the dye molecule less hydrophilic, which in turn increases the aggregation of labeled biomolecules in aqueous media and increases the tendency for forming non-fluorescent dye-dye dimers.
Therefore, there is a need for xanthene class fluorescent dyes that do not undergo formation of non-fluorescent lactone/lactam and, at the same time, do not undergo hydrolysis in aqueous media.